Enantioselective synthesis of (S)-phenylephrine by recombinant Escherichia coli cells expressing the short-chain dehydrogenase/reductase gene from Serratia quinivorans BCRC 14811
Introduction
Phenylephrine (PE) is a sympathomimetic, which is widely used as a nasal decongestant in common cold and flu medicines [1]. Other sympathomimetics, including phenylpropanolamine, pseudoephedrine (PDE) and ephedrine, are also used in cough and cold medicines [2]. However, the over-the-counter (OTC) and prescription drug products containing phenylpropanolamine were no longer recommended for use by the Food and Drug Administration in the year 2000 because these drugs were associated with an increased risk of hemorrhagic stroke [3]. PDE and PE are now commonly used worldwide as systemic nasal decongestants. Because PDE is easily abused for the production of methamphetamine [4], [5], the Combat Methamphetamine Epidemic Act in the USA banned OTC medicines containing PDE in 2005 to avoid the illicit conversion of PDE into methamphetamine [6]. This restriction imposed on PDE has led to a need for PE in common cold medicines.
PE contains one chiral carbon atom in the Cα of the side chain, and the R enantiomer of PE exhibits more potency than the S enantiomer for the activation of α1-adrenergic receptors [7]. PE can be produced by various methods involving ring opening of 3-benzyl-oxystyrene epoxide [8], Curtius rearrangement of a β-hydroxy acid azide [9] and reduction of a mandelamide [10]. PE produced from these methods is a racemic mixture; therefore, asymmetric hydrogenation methods have been developed for the production of (R)-PE [11], [12], [13], [14]. However, these methods require high pressure, high temperature and several environmentally unfriendly organic solvents.
We have cloned an amino alcohol dehydrogenase gene (RE_AADH) from Rhodococcus erythropolis BCRC 10909 into Escherichia coli, and the cells expressing RE_AADH were used for the conversion of 1-(3-hydroxyphenyl)-2-(methylamino) ethanone (HPMAE) to (S)-PE [15], which can be further converted to (R)-PE by the Walden inversion reaction. However, RE_AADH used NADPH as cofactor, and only limited production of PE from HPMAE was achieved. In this study, we have described the cloning of a short-chain dehydrogenase/reductase gene from Serratia quinivorans BCRC 14811 and expression in E. coli BL21 (DE3). The biochemical properties of recombinant SQ_SDR were characterized. We found that the SQ_SDR enzyme could utilize both NADH and NADPH as a cofactor for its catalytic activity. The yield and productivity of the conversion of HPMAE to PE by recombinant E. coli expressing SQ_SDR were also evaluated.
Section snippets
Materials
All solvents were LC grade and purchased from Merck (Darmstadt, Germany) and Sigma–Aldrich (St. Louis, MO, USA). Restriction enzymes were obtained from New England Biolabs (Ipswich, MA, USA). DNA polymerase, ExTaq, and T4 DNA ligase were purchased from TaKaRa (Tokyo, Japan). Culture media were obtained from Becton, Dickinson and Company (Sparks, MD, USA).
Bacterial strains, plasmid and culture conditions
E. coli BL21 (DE3) was cultivated in Luria-Bertani (LB) medium at 37 °C. All bacterial strains were obtained from the Bioresource Collection
Screening of bacterial strains for the conversion of HPMAE to PE
In a previous report, we found that R. erythropolis BCRC 10909 converted just detectable levels of HPMAE to (S)-PE and that E. coli NovaBlue expressing RE_AADH from R. erythropolis BCRC 10909 converted HPMAE to PE with limited productivity [15]. Therefore, 13 strains of bacteria were screened for their ability to convert HPMAE to PE. As shown in Table 1, S. quinivorans BCRC 14811 was the most promising bacterium in this test, with a yield of 36.8%.
Cloning of the gene responsible for HPMAE reduction from S. quinivorans BCRC 14811
The genomic sequence of S. quinivorans
Conclusion
The SQ_SDR gene was cloned from S. quinivorans BCRC 14811 and functionally expressed in E. coli BL21 (DE3). The cell enzyme activity for the recombinant strain was 3.23 U/g wet cells. The recombinant E. coli strain expressing NADH-preferring SQ_SDR exhibited a much higher conversion yield and productivity for the conversion of HPMAE to (S)-PE than did our previous strain, E. coli NovaBlue expressing NADPH-dependent RE_AADH. We demonstrated that the SDR enzyme with its high catalytic activity and
Acknowledgements
This work was supported by the research grants NSC95-2313-B-005-005 and NSC96-2628-B-005-002-MY3 from the National Science Council, Taiwan.
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